A team of researchers from China has successfully utilized an innovative form of the genome-editing technique CRISPR to insert a new gene into the cow genome, rendering the animals much more resistant to tuberculosis. They used a novel version of the CRISPR system called CRISPR/Cas9n to successfully insert a tuberculosis resistance gene into the cow genome.

The Process

In this study, the investigators inserted the NRAMP1 gene into the genome of bovine fetal fibroblasts—cells derived from female dairy cows—using the CRISPR/Cas9n technology. These cells were then used as donor cells in a process called somatic cell nuclear transfer (therapeutic cloning), where the nucleus of a donor cell carrying the new gene is inserted into an egg cell, known as an ovum, from a female cow.

Next, the ova were nurtured in the lab into embryos before being transferred into mother cows for a normal pregnancy cycle. As a control, the experiments were also conducted using the standard CRISPR/Cas9 technology as a comparison. A total of 11 calves with new genes inserted using CRISPR were able to be assessed for resistance to tuberculosis and any off-target genetic effects.

Genetic analysis of the calves revealed that NRAMP1 had successfully integrated into the genetic code at the targeted region in all of the calves. None of the calves that had the gene inserted using the new CRISPR/Cas9n technology had any detectable off-target effects, whereas all of the calves with the gene inserted with previously used techniques for CRISPR/Cas9 did.

Remarkably, when the calves were exposed to Mycobacterium bovis, the bacterium that causes bovine tuberculosis, the researchers found that transgenic animals showed an increased resistance to the bacterium measured by standard markers of infection in a blood sample. Additionally, they found that white blood cells taken from the calves were much more resistant to M. bovis exposure in laboratory tests.

Background

CRISPR technology has become widely used in the laboratory in recent years as it is an accurate and relatively easy way to modify the genetic code. The technology allows scientists to go in and essentially snip and tuck genes from one organism to another to enhance them in some way, and it’s already been done with pigs, fish, mice, and mosquitos, as well as human embryos.

However, sometimes unintentional changes to the genetic code occur as an off-target effect, so finding ways to reduce these is a priority for genomics research.

Significance

With this, the researchers have successfully developed live cows carrying increased resistance to tuberculosis. Importantly, this method produced no off-target effects on the cow genetics, meaning that the CRISPR technology employed may be better suited to producing transgenic livestock with purposefully manipulated genetics.